Project Summary Bidirectional transport of cargo in neurons is required for their growth and function. The opposite-polarity cytoskeletal motors, kinesin and dynein, are responsible for driving transport toward the synaptic terminals and back to the cell center, respectively. Bidirectional transport necessarily involves a complex web of regulatory factors from the inherent properties of the motors themselves to structural changes in the microtubule tracks they walk on. Breakdown in the neuronal transport machinery is implicated in several neurodegenerative diseases, including Alzheimer?s, Parkinson?s, Huntington?s and amyotrophic lateral sclerosis (ALS). Unfortunately, the underlying mechanism of bidirectional transport is not known due to experimental limitations that have prevented direct observation of motors working in an ensemble. A widely accepted model is ?tug-of- war?, wherein teams of kinesin and dynein pull against each other and the winning team determines the direction of travel. However, the tug-of-war model does not account for motor-motor coordination and other possible regulatory factors. The long-term goal of this project is to establish an in vitro method for direct observation of motor ensembles that can be easily adapted to understand all facets of bidirectional transport. The proposed method combines gold nanoparticle tracking via Interferometric Scattering (iSCAT) microscopy with DNA origami to create a versatile multi-motor assay that enables precise visualization of a single motor or overall cargo displacement during collective transport. The short-term goal of this project is to better understand the motility of adaptor-activated mammalian dynein (DDB) as it transports cargo in a pair with a cooperative or antagonistic partner. Aim 1 will investigate how the stepping of DDB is adapted for bidirectional movement by tracking the motor domain as it steps with another DDB or kinesin-1. The results will shed light on why dynein works better in a team than kinesin-1, and how dynein remains actively engaged with the cargo during kinesin-driven transport. Aim 2 will investigate how the motility properties of the kinesin-2 and kinesin-3 families affect their ability to compete against DDB in tug-of-war by tracking the motility of DDB-kinesin-2 and DDB-kinesin-3 pairs. The results will provide insight into how transport is regulated by the population of kinesin families that are bound to the cargo, and how motility properties can influence kinesin-dynein coordination. Execution of the proposed experiments will require extensive knowledge in DNA origami design, baculovirus expression of complex proteins, analysis of high-resolution tracking data, and construction and optimization of a high-resolution microscope. Therefore, the overall outcome of the project will not only be significant advancement in understanding of the connection between single motor stepping and collective transport in the cell, but also excellent training in cutting-edge single molecule microscopy techniques.